The mycobacteria are a genus of bacteria which are acid-fast, non-motile, gram-positive rods. The genus comprises several species which include, but are not limited to, Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. microti, M. scrofulaceum, M. paratuberculosis and M. tuberculosis. Certain of these organisms are the causative agents of disease. For the first time since 1953, cases of mycobacterial infections are increasing in the United States. Of particular concern is tuberculosis, the etiological agent of which is M. tuberculosis. Many of these new cases are related to the AIDS epidemic, which provides an immune compromised population which is particularly susceptible to infection by mycobacteria. Other mycobacterial infections are also increasing as a result of the increase in numbers of immune compromised patients. Mycobacterium avium, Mycobacterium kansasii and other non-tuberculosis mycobacteria are found as opportunistic pathogens in HIV infected and other immune compromised patients.
Conventional diagnosis of mycobacterial infections relies on acid-fast staining and cultivation of the organism, followed by biochemical assays. These procedures are time-consuming, and a typical diagnosis using conventional culture methods can take as long as six weeks. Automated culturing systems such as the BACTEC.TM. system (Becton Dickinson Microbiology Systems, Sparks, Md.) can decrease the time for diagnosis to one to two weeks. However, there is still a need to reduce the time required for diagnosing mycobacterial infections to less than a week, preferably to about one day. Oligonucleotide probe based assays such as Southern hybridizations or dot blots are capable of returning a rapid result (i.e., in one day or less). Assays based on amplification of nucleic acids are usually more sensitive and may provide even more rapid results, often within hours. For diagnosis of mycobacterial infections such methods require development of oligonucleotide probes or primers which are specific for the genus Mycobacterium or specific for a particular species of mycobacteria if specific identification of the organism is desired.
Conventional laboratory identification of Mycobacterium kansasii is based upon biochemical testing and determination of growth characteristics. These include catalase production, urease activity, TWEEN hydrolysis, nitrate reduction and the ability of the bacterium to produce pigment when exposed to light (photochromogenicity). Because several other mycobacterial species exhibit a similar biochemical profile, photochromogenicity is generally relied upon for conclusive identification of Mycobacterium kansasii. However, determination of photochromogenicity requires a pure culture of the organism and this phenotype can be variable, subjective and difficult to determine reliably. For these reasons, there have been attempts to identify Mycobacterium kansasii by species-specific hybridization or nucleic acid amplification using oligonucleotide probes. Z. H. Huang, et al. (1991. J. Clin. Microbiol. 29, 2125-2129) have reported a DNA probe obtained from a genomic library with a degree of species-specificity for Mycobacterium kansasii. This clone (pMK 1-9) showed some cross-hybridization with other species, including M. gastri, and did not detect a genetically distinct subgroup ofM. kansasii. The nucleotide sequence of pMK1-9 was not reported, nor was the gene from which it may have been derived identified. B. C. Ross, et al. (1992. J. Clin. Microbiol. 30, 2930-2933) also reported identification of genetically distinct subspecies of M. kansasii using the pMK1-9 probe, a 65 kDa antigen gene probe and a commercial DNA probe test employing probes which specifically hybridize to rRNA (ACCU-PROBE, Gen-Probe, San Diego, Calif.). T. Rogall, et al. (1990. J. Gen. Microbiol. 136, 1915-1920) used the 16S rRNA sequence in a polymerase chain reaction (PCR) based sequencing strategy for identification of mycobacterial species. However, these primers could not be used to differentiate M. gastri from M. kansasii because the 16S rRNA sequence from these two species is identical in spite of their differing phenotypic characteristics. Similar studies have been published by B. B oddinghaus, et al. (1990. J. Clin. Microbiol. 28, 1751-1759), who reported oligonucleotides derived from 16S rRNA sequences which are specific for the M. tuberculosis group, i.e., M. avium-M. paratuberculosis, and M. intracellulare. M. Yang, et al. (1993. J. Clin. Microbiol. 31, 2769-2772) have reported isolation of a sequence from a clinical isolate which, when used as a hybridization probe, exhibits M. kansasii species-specificity. This probe (p6123) hybridized to all M. kansasii strains tested, including the subgroup which is pMK1-9 negative. P. A. Spears (U.S. Pat. No. 5,500,341) describes M. kansasii-specific primers derived from the region of p6123 between nucleotides 51 and 220.
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. Amplification primers are typically about 10-75 nucleotides in length, preferably about 15-50 nucleotides in length. The total length of an amplification primer for SDA is typically about 25-50 nucleotides. The 3' end of an SDA amplification primer (the target binding sequence) hybridizes at the 3' end of the target sequence. The target binding sequence is about 10-25 nucleotides in length and confers hybridization specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease 5' to the target binding sequence. The recognition site is for a restriction endonuclease which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by G. Walker, et al. (1992. PNAS 89:392-396 and 1992 Nucl. Acids Res. 20:1691-1696). The nucleotides 5' to the restriction endonuclease recognition site (the "tail") function as a polymerase repriming site when the remainder of the amplification primer is nicked and displaced during SDA. The repriming function of the tail nucleotides sustains the SDA reaction and allows synthesis of multiple amplicons from a single target molecule. The tail is typically about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified to obtain the desired T.sub.m for hybridization. As the target binding sequence is the portion of a primer which determines its target-specificity, for amplification methods which do not require specialized sequences at the ends of the target the amplification primer generally consists essentially of only the target binding sequence. For amplification methods which require specialized sequences appended to the target other than the nickable restriction endonuclease recognition site and the tail of SDA (e.g., an RNA polymerase promoter for 3SR, NASBA or transcription based amplification), the required specialized sequence may be linked to the target binding sequence using routine methods for preparation of oligonucletoides without altering the hybridization specificity of the primer.
A bumper primer or external primer is a primer used to displace primer extension products in isothermal amplification reactions. The bumper primer anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream amplification primer and its extension product.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies serve as amplifiable targets by virtue of the fact that they contain copies of the sequence to which the amplification primers hybridize.
Copies of the target sequence which are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the copy of a target sequence produced by hybridization of a primer and extension of the primer by polymerase using the target sequence as a template.
The term species-specific refers to detection, amplification or oligonucleotide hybridization in a species of organism or a group of related species without substantial detection, amplification or oligonucleotide hybridization in other species of the same genus or species of a different genus.
The term assay probe refers to any oligonucleotide used to facilitate detection or identification of a nucleic acid. For example, in the present invention, assay probes are used for detection or identification of Mycobacterium kansasii nucleic acids. Detector probes, detector primers, capture probes and signal primers as described below are examples of assay probes.